Recombinant Vibrio vulnificus Putative reductase VV1437 (VV1437)

What is VV1437 and what organism does it come from?

VV1437 is a putative reductase protein derived from Vibrio vulnificus strain YJ016, an opportunistic human pathogen found in coastal marine environments worldwide . This bacterium is responsible for the majority of seafood-related deaths in the United States, causing severe infections with mortality rates exceeding 50% . The protein is classified as EC 1.3.1.-, indicating its likely function as a reductase involved in metabolic processes, and has been assigned the UniProt accession number Q7MLJ0 . In target name nomenclature, VV1437 is also referred to as fabV1, suggesting a potential role in fatty acid biosynthesis pathways that are essential for bacterial membrane formation and cellular integrity .

What are the optimal storage conditions for recombinant VV1437?

The optimal storage conditions for recombinant VV1437 depend on its formulation state . For the liquid form, the recommended storage temperature is -20°C to -80°C, providing a shelf life of approximately 6 months . The lyophilized (freeze-dried) form offers extended stability with a shelf life of approximately 12 months when stored at the same temperature range .

For working with the protein, several handling considerations are important: repeated freezing and thawing should be avoided as this can compromise protein integrity and activity; working aliquots may be stored at 4°C for up to one week; and for long-term storage, the protein should be reconstituted and supplemented with glycerol (5-50% final concentration, with 50% being the default recommendation) before aliquoting and freezing .

The shelf life of recombinant proteins like VV1437 is influenced by multiple factors, including storage state, buffer ingredients, storage temperature, and the intrinsic stability of the protein itself . Proper storage is critical for maintaining enzymatic activity and structural integrity for experimental applications.

How is Vibrio vulnificus related to human disease?

Vibrio vulnificus is an opportunistic human pathogen responsible for the overwhelming majority of seafood-related deaths in the United States . This bacterium naturally occurs in coastal marine environments worldwide and has been isolated from water, sediments, and various seafood including shrimp, fish, oysters, and clams .

Consumption of raw seafood, particularly oysters, containing V. vulnificus can lead to a severe, fulminant systemic infection characterized by fever, chills, nausea, hypotensive septic shock, and the formation of secondary lesions on the extremities . This primary septicemia is extremely lethal, with an average mortality rate exceeding 50% .

Analysis of 459 U.S. cases reported between 1992 and 2007 revealed that 51.6% of the patients died, with a notable gender disparity where 85.6% of cases occurred in males . Among 180 cases from 2002 to 2007 with available data, 92.8% of patients had consumed raw oysters prior to symptom onset, and 95.3% had preexisting health conditions, suggesting that individuals with compromised immune systems or underlying diseases are particularly vulnerable to this pathogen .

What is the predicted functional role of VV1437 in Vibrio vulnificus metabolism?

Based on its EC classification (1.3.1.-) and the "fabV1" target name, VV1437 likely functions as a reductase involved in fatty acid biosynthesis pathways . The EC 1.3.1.- designation indicates it belongs to the oxidoreductase family, specifically acting on the CH-CH group of donors with NAD+ or NADP+ as acceptor.

The sequence characteristics of VV1437 suggest it contains NAD(P)H binding domains typical of reductases in the fatty acid synthesis pathway . In bacteria, these enzymes catalyze the reduction of enoyl-ACP (acyl carrier protein) intermediates, a critical step in the fatty acid elongation cycle. This process is essential for membrane phospholipid biosynthesis and thus cellular integrity and survival.

In the context of V. vulnificus pathogenesis, metabolic enzymes like VV1437 may play indirect roles in virulence by ensuring bacterial fitness during infection. While not directly comparable to established virulence factors like RtxA1 toxin , metabolic enzymes can be crucial for bacterial adaptation to host environments. Understanding VV1437's precise role could reveal connections between V. vulnificus metabolism and pathogenicity, potentially identifying new targets for antimicrobial interventions.

How might experimental approaches elucidate interactions between VV1437 and other V. vulnificus proteins?

To investigate potential interactions between VV1437 and other V. vulnificus proteins, researchers can employ multiple complementary experimental approaches:

• Co-immunoprecipitation (Co-IP): Using antibodies against VV1437 to pull down protein complexes from V. vulnificus lysates, followed by mass spectrometry to identify interacting partners. This approach can reveal both stable and transient protein-protein interactions under native conditions.

• Bacterial two-hybrid systems: Modified versions of yeast two-hybrid systems adapted for bacterial proteins can screen for binary interactions between VV1437 and potential partners.

• Proximity-dependent biotin labeling: Techniques like BioID or APEX2, where VV1437 is fused to a biotin ligase that biotinylates proximal proteins, can identify proteins in close spatial proximity in living bacteria.

• Cross-linking mass spectrometry: Chemical cross-linking of proteins followed by mass spectrometry analysis can capture transient interactions and provide structural information about the interaction interface.

• Genetic interaction screens: Systematic creation of double mutants combining VV1437 mutations with mutations in other genes can reveal functional relationships through synthetic lethality or suppressor effects.

Understanding protein interactions is particularly relevant when considering how metabolic enzymes like VV1437 might be integrated with virulence systems such as the RtxA1 toxin complex . Such interactions could reveal unexpected roles for VV1437 in regulating or supporting virulence factor production and activity during infection.

How can power analysis improve experimental design in VV1437 functional studies?

Power analysis is a critical statistical tool that helps researchers determine the appropriate sample size needed to detect effects of a given magnitude with specified confidence levels3. For VV1437 functional studies, proper power analysis can prevent both type I errors (false positives) and type II errors (false negatives), ensuring experimental rigor and reproducibility.

When designing enzyme kinetic studies for VV1437, power analysis would help determine the number of replicate measurements needed to reliably detect differences in enzyme activity under various conditions. The sample size (n) can be calculated using the formula:

$n = \frac{2(Z_{\alpha/2} + Z_{\beta})^2 \sigma^2}{\Delta^2}$

Where:

• $Z_{\alpha/2}$ is the critical value for significance level α (1.96 for α = 0.05)

• $Z_{\beta}$ is the critical value for power 1-β (0.84 for 80% power)

• $σ$ is the standard deviation of the measurements

• $Δ$ is the minimum effect size of interest

For example, when comparing wild-type VV1437 activity with a site-directed mutant, preliminary data might suggest an expected 30% reduction in activity with a standard deviation of 15%. To detect this difference with 80% power at α = 0.05, the required sample size would be calculated using the above formula.

Power analysis is equally important for in vivo studies examining the role of VV1437 in V. vulnificus virulence, where sample sizes must be sufficient to detect biologically meaningful differences in outcomes like bacterial load or host response while minimizing animal use3. This approach ensures both scientific validity and ethical experimental design.

What challenges exist in distinguishing the role of VV1437 from other reductases in V. vulnificus?

Distinguishing the specific role of VV1437 from other reductases in V. vulnificus presents several methodological challenges:

• Functional redundancy: Bacterial genomes often contain multiple genes encoding enzymes with overlapping substrate specificities. For instance, other NAD(P)H-dependent reductases in V. vulnificus may compensate for VV1437 function when it is deleted, masking phenotypic effects in knockout studies.

• Substrate ambiguity: The EC classification 1.3.1.- encompasses a broad range of reductases . Without definitive biochemical characterization, determining the specific physiological substrate(s) of VV1437 remains challenging.

• Conditional expression: VV1437 may only be significantly expressed under specific environmental conditions relevant to infection, making laboratory assessment of its function difficult under standard culturing conditions.

• Protein purification issues: Recombinant expression may not fully recapitulate the native conformation or post-translational modifications of VV1437, potentially affecting in vitro activity assays.

To address these challenges, researchers can employ several strategies:

• Complementary approaches: Combining genetic (knockout/complementation), biochemical (in vitro activity), and systems-level (transcriptomics/proteomics) approaches to build a comprehensive understanding of VV1437 function.

• Conditional expression systems: Using controlled expression systems to study VV1437 function under different environmental conditions.

• Specific inhibitors: Developing selective chemical probes that target VV1437 without affecting other reductases.

• Comparative genomics: Analyzing VV1437 conservation across Vibrio species and correlating sequence variations with phenotypic differences.

These strategies would help delineate the specific contribution of VV1437 to V. vulnificus metabolism and potentially pathogenesis, despite the challenges of functional overlap with other reductases.

What are the optimal conditions for reconstituting recombinant VV1437 protein?

The optimal reconstitution procedure for recombinant VV1437 involves several critical steps to ensure protein stability and activity :

• Initial preparation: Prior to opening, the vial should be briefly centrifuged to collect the protein at the bottom, minimizing product loss during handling .

• Reconstitution medium: The protein should be reconstituted in deionized sterile water to a concentration ranging from 0.1-1.0 mg/mL . The use of sterile water helps prevent microbial contamination that could degrade the protein or interfere with downstream applications.

• Cryoprotection for storage: For long-term storage, glycerol should be added to a final concentration of 5-50%, with 50% being the recommended default . Glycerol acts as a cryoprotectant, preventing ice crystal formation that can denature proteins during freeze-thaw cycles.

• Aliquoting strategy: The reconstituted protein should be divided into small aliquots to avoid repeated freeze-thaw cycles of the entire stock, as this is specifically not recommended for VV1437 .

• Storage conditions: Long-term storage should be at -20°C to -80°C, providing a shelf life of approximately 6 months for liquid formulations and 12 months for lyophilized preparations . Working aliquots can be stored at 4°C for up to one week .

This reconstitution protocol is designed to maintain the structural integrity and enzymatic activity of VV1437, which is crucial for subsequent experimental applications such as activity assays, structural studies, or antibody production. The specific recommendations reflect general principles of protein handling optimized for this particular recombinant protein.

How can researchers design activity assays for VV1437 as a putative reductase?

Designing activity assays for VV1437 requires consideration of its putative function as a reductase (EC 1.3.1.-) potentially involved in fatty acid biosynthesis . A comprehensive approach would include:

• Substrate selection: Based on the "fabV1" designation, potential substrates include enoyl-ACP intermediates or analogous compounds such as crotonyl-CoA, trans-2-hexenoyl-CoA, or commercially available model substrates like trans-2-hexenal.

• Cofactor requirements: As a putative oxidoreductase, VV1437 likely requires NAD(P)H as an electron donor. Assays should include both NADH and NADPH to determine cofactor preference. Activity can be monitored spectrophotometrically by measuring the decrease in absorbance at 340 nm as NAD(P)H is oxidized.

• Reaction conditions optimization:

  Parameter	Range to Test	Methodology
  pH	6.0-9.0	Buffer series: MES (pH 6.0-6.5), PIPES (pH 6.5-7.5), HEPES (pH 7.0-8.0), Tris (pH 7.5-9.0)
  Temperature	25-45°C	Temperature-controlled spectrophotometer
  Ionic strength	0-500 mM NaCl	Salt titration in reaction buffer
  Divalent cations	0-10 mM Mg²⁺, Mn²⁺, Ca²⁺	Metal ion supplementation

• Kinetic parameter determination: Varying substrate concentrations (typically 0.1-10× Km) to determine Michaelis-Menten kinetic parameters:

  • Km (substrate affinity)

  • kcat (catalytic rate constant)

  • kcat/Km (catalytic efficiency)

• Controls and validation:

  • Negative controls: Heat-inactivated enzyme, no substrate, no enzyme

  • Positive controls: Related characterized reductases

  • Specificity controls: Testing structurally similar non-substrate compounds

• Data analysis: Non-linear regression analysis using enzyme kinetics software to fit data to appropriate models (Michaelis-Menten, allosteric, inhibition models as applicable).

This methodological framework provides a systematic approach to characterizing the enzymatic properties of VV1437, which is essential for understanding its biochemical function and potential role in V. vulnificus metabolism.

What approaches can be used to study the role of VV1437 in Vibrio vulnificus virulence?

To investigate the potential role of VV1437 in Vibrio vulnificus virulence, researchers can employ multiple complementary approaches:

• Genetic manipulation studies:

  • Generate VV1437 knockout mutants using targeted mutagenesis

  • Create conditional expression strains where VV1437 levels can be modulated

  • Develop complementation strains to confirm phenotypes are specifically due to VV1437 disruption

  • Introduce site-directed mutations in catalytic residues to distinguish enzymatic activity from potential structural roles

  This genetic approach has been successfully used for studying other V. vulnificus factors such as RtxA1 toxin .

• Comparative virulence assessment:

  • Compare wild-type and VV1437-deficient strains in established infection models

  • Assess parameters including cytotoxicity, invasion capacity, and in vivo survival

  • Measure virulence markers such as LD50 values (as done with RtxA1 studies)

  • Evaluate tissue distribution and bacterial load in animal models

• Expression analysis under infection-relevant conditions:

  • Examine VV1437 expression under conditions mimicking the host environment (serum exposure, iron limitation, various pH values)

  • Compare expression patterns with known virulence factors like RtxA1

  • Use reporter gene fusions to monitor expression in real-time during infection

  • Perform RNA-seq to identify co-regulated genes

• Biochemical characterization in host-relevant contexts:

  • Assess VV1437 activity in the presence of host factors

  • Investigate potential post-translational modifications during infection

  • Determine if host conditions affect substrate specificity or kinetic parameters

• Systems-level analysis:

  • Conduct proteomics and metabolomics analyses comparing wild-type and VV1437-mutant strains

  • Map metabolic pathways potentially affected by VV1437 activity

  • Identify potential connections between VV1437-dependent metabolic processes and established virulence mechanisms

These methodological approaches would provide comprehensive insights into whether and how VV1437 contributes to V. vulnificus pathogenicity, building on established protocols used for characterizing virulence factors like RtxA1 .

How can structural analysis of VV1437 inform functional studies?

Structural analysis of VV1437 can provide crucial insights that inform and guide functional studies through multiple approaches:

What statistical approaches are appropriate for analyzing VV1437 enzyme kinetics data?

When analyzing enzyme kinetics data for VV1437, several statistical approaches are appropriate depending on the experimental design and research questions:

• Michaelis-Menten kinetics analysis:

  • Non-linear regression to fit the Michaelis-Menten equation:
    $v = \frac{V_{max} \cdot [S]}{K_m + [S]}$

  • Extract kinetic parameters (Km, Vmax) with 95% confidence intervals

  • Use residual analysis to assess goodness of fit

  • Compare different enzyme kinetic models (Michaelis-Menten, substrate inhibition, allosteric) using AIC (Akaike Information Criterion) or BIC (Bayesian Information Criterion)

• Comparative kinetics analysis:

  • ANOVA with post-hoc tests (Tukey's HSD, Bonferroni correction) to compare kinetic parameters across experimental conditions

  • t-tests for pairwise comparisons of wild-type vs. mutant enzymes

  • Determine appropriate sample sizes through power analysis3

• Inhibition studies analysis:

  • Dixon plots and Lineweaver-Burk plots to distinguish inhibition mechanisms

  • Global fitting of multiple datasets to different inhibition models

  • Determination of inhibition constants (Ki) with confidence intervals

• Environmental factor effects:

  • Multiple regression analysis for multifactorial experiments

  • Response surface methodology to model effects of pH, temperature, and ionic strength on enzyme activity

  • Principal component analysis to identify major factors affecting enzyme behavior

• Data visualization approaches:

  • Residual plots to assess model adequacy

  • Bootstrap resampling to generate robust confidence intervals

  • Heat maps for visualizing activity across multiple conditions

A rigorous approach would include:

• Data cleaning and outlier detection

• Normality testing of distributions

• Application of appropriate statistical model with consideration of sample size requirements from power analysis3

• Parameter estimation with confidence intervals

• Model validation and diagnostics

• Interpretation in the context of biological function

How can researchers correlate VV1437 activity with Vibrio vulnificus pathogenicity?

Correlating VV1437 activity with Vibrio vulnificus pathogenicity requires a multifaceted approach that connects molecular function to virulence phenotypes:

• Comparative genomic analysis:

  • Analyze VV1437 sequence variations across clinical and environmental isolates

  • Correlate polymorphisms with virulence phenotypes

  • Examine whether VV1437 variants cluster with known pathogenicity markers, similar to observed patterns with other virulence genes in V. vulnificus

• Expression correlation studies:

  • Measure VV1437 expression levels across strains with different virulence potentials

  • Analyze co-expression patterns with established virulence factors like RtxA1

  • Use transcriptomic data to identify conditions that co-regulate VV1437 with virulence genes

• Phenotypic characterization of VV1437 mutants:

  • Compare cytotoxicity of wild-type and VV1437-deficient strains (similar to approaches used for RtxA1)

  • Assess bacterial survival in human serum

  • Measure in vivo virulence using established animal models

  • Evaluate key virulence phenotypes such as biofilm formation, motility, and stress resistance

• Biochemical pathway analysis:

  • Determine whether VV1437 enzymatic activity affects membrane composition

  • Investigate if VV1437-dependent metabolic changes influence toxin production or secretion

  • Assess whether VV1437 activity affects bacterial fitness under host-like conditions

• Integration of multiple data types:

  Data Type	Analytical Approach	Correlation to Pathogenicity
  Genetic variation	SNP analysis, phylogenetics	Association with clinical vs. environmental strains
  Expression data	RNA-seq, qPCR	Co-regulation with virulence factors
  Metabolic profiles	Metabolomics, flux analysis	Impact on virulence-associated pathways
  Virulence phenotypes	Infection models, cytotoxicity	Direct measures of pathogenicity

This integrated approach would establish whether VV1437 contributes to V. vulnificus pathogenicity either directly or indirectly through metabolic functions that support virulence. The analysis would build upon established methodologies used for characterizing other virulence factors, such as RtxA1, which has been shown to significantly impact cytotoxicity and lethality .

What bioinformatic tools are most appropriate for analyzing VV1437 in the context of bacterial metabolism?

To analyze VV1437 in the context of bacterial metabolism, researchers should utilize a combination of bioinformatic tools that address different aspects of protein function and metabolic integration:

• Protein function prediction tools:

  • InterProScan: Identifies protein domains, families, and functional sites

  • PFAM: Detects conserved protein domains

  • PROSITE: Identifies functional motifs and protein signatures

  • ConSurf: Maps evolutionary conservation onto protein structures

  • I-TASSER/AlphaFold2: Generates 3D structural models for function prediction

• Metabolic pathway analysis tools:

  • KEGG Pathway: Maps enzymes to metabolic pathways

  • BioCyc/MetaCyc: Provides detailed metabolic pathway information

  • BRENDA: Offers comprehensive enzyme information

  • STRING: Identifies protein-protein interaction networks

  • RAST: Annotates bacterial genomes with metabolic functions

• Comparative genomics approaches:

  • OrthoMCL/OrthoFinder: Identifies orthologous proteins across bacterial species

  • Clustal Omega: Performs multiple sequence alignments to identify conserved regions

  • BLAST: Compares VV1437 sequence against diverse bacterial genomes

  • Roary: Analyzes pan-genome to identify core and accessory genes

• Regulatory network analysis:

  • RegulonDB: Identifies potential regulatory elements

  • MEME Suite: Discovers motifs in DNA sequences that might regulate VV1437

  • TFBScan: Predicts transcription factor binding sites

• Integrated analysis workflow:

  Analysis Stage	Bioinformatic Tools	Expected Insights
  Sequence analysis	BLAST, Clustal Omega, InterProScan	Evolutionary relationships, conserved domains
  Structural prediction	AlphaFold2, PyMOL, ConSurf	Active site architecture, substrate binding
  Metabolic context	KEGG, BioCyc, BRENDA	Position in fatty acid biosynthesis pathways
  Regulatory analysis	MEME, RegulonDB	Potential co-regulation with virulence factors
  Systems integration	STRING, Cytoscape	Protein interaction networks, pathway visualization

• Genome-scale metabolic modeling:

  • COBRApy: Constructs genome-scale metabolic models

  • OptFlux: Performs flux balance analysis

  • MEMOTE: Tests and validates metabolic models

These bioinformatic tools would provide a comprehensive understanding of VV1437's role in V. vulnificus metabolism, potentially revealing connections between metabolic functions and virulence mechanisms that could be experimentally validated. This systems biology approach is particularly valuable given the complex relationship between metabolism and pathogenicity in bacterial infections.

How can researchers interpret contradictory results in VV1437 functional studies?

When faced with contradictory results in VV1437 functional studies, researchers should employ a systematic approach to resolution that considers multiple factors:

This systematic approach recognizes that contradictory results often reflect biological complexity rather than experimental error, and may reveal important insights about condition-specific functions or strain-specific adaptations of VV1437 in V. vulnificus metabolism and pathogenicity.

What are the future research directions for VV1437 studies?

Future research on VV1437 should focus on several promising directions that would enhance understanding of this putative reductase and its potential role in Vibrio vulnificus biology:

• Comprehensive biochemical characterization:

  • Definitive substrate identification through systematic screening of potential metabolites

  • Detailed kinetic analysis under physiologically relevant conditions

  • Determination of three-dimensional structure through X-ray crystallography or cryo-EM

  • Elucidation of the catalytic mechanism through site-directed mutagenesis

• Genetic and phenotypic analysis:

  • Construction of clean deletion mutants and complementation strains

  • Phenotypic characterization under various environmental conditions

  • Assessment of VV1437 contribution to stress resistance

  • Investigation of potential moonlighting functions beyond primary enzymatic activity

• Connection to pathogenesis:

  • Evaluation of VV1437 role in virulence using animal infection models

  • Investigation of potential interactions with established virulence factors like RtxA1

  • Examination of VV1437 expression during different phases of infection

  • Comparison of VV1437 variants across clinical and environmental isolates

• Metabolic integration:

  • Metabolomic analysis of VV1437 mutants to identify affected pathways

  • Investigation of how VV1437 activity affects membrane composition

  • Assessment of metabolic adaptation in host environments

  • Development of genome-scale metabolic models incorporating VV1437 function

• Therapeutic potential:

  • Screening for specific inhibitors of VV1437

  • Assessment of inhibitor effects on bacterial growth and virulence

  • Evaluation of VV1437 as a potential vaccine antigen

  • Investigation of synergistic effects between VV1437 inhibition and conventional antibiotics

These research directions would contribute to a more complete understanding of VV1437's biological significance in V. vulnificus and potentially reveal new strategies for combating this deadly pathogen. Future work should build upon the established methodologies used to characterize other factors in V. vulnificus, while incorporating emerging technologies for more comprehensive analysis.

How does understanding VV1437 contribute to broader knowledge of bacterial metabolism and virulence?

Understanding VV1437 contributes to broader knowledge of bacterial metabolism and virulence through several significant dimensions:

• Bridging metabolism and virulence:

  • VV1437, as a putative reductase likely involved in fatty acid biosynthesis , represents an important link between core metabolism and pathogenicity

  • Its study can illuminate how metabolic enzymes indirectly support virulence factor production and function

  • This research contributes to the growing field investigating metabolism as a virulence determinant

• Evolutionary insights:

  • Comparative analysis of VV1437 across Vibrio species can reveal how metabolic enzymes evolve in pathogenic contexts

  • Understanding whether VV1437 variants correlate with virulence potential could demonstrate how metabolic adaptations contribute to pathogen evolution

  • This connects to broader patterns observed in V. vulnificus strain diversity and virulence potential

• Systems biology perspective:

  • VV1437 research exemplifies how single protein studies can be integrated into whole-cell models

  • Mapping its interactions within metabolic networks provides templates for understanding complex bacterial systems

  • This contributes to comprehensive models of how bacteria coordinate metabolism and virulence

• Therapeutic strategy development:

  • Characterizing VV1437 may reveal metabolic vulnerabilities that could be exploited for antimicrobial development

  • This adds to growing strategies targeting metabolic enzymes rather than classical virulence factors

  • Such approaches may be less susceptible to resistance development than conventional antibiotics

• Methodological advancement:

  • Developing robust approaches to study VV1437 contributes techniques applicable to other bacterial enzymes

  • The integration of power analysis in experimental design3 with molecular techniques demonstrates rigorous research methodology

  • Multi-omics integration strategies developed for VV1437 research can serve as templates for other bacterial systems